Sperm Motility Characteristics Research Articles

Cryopreservation of bison semen without exogenous protein in extender and its fertility potential in vitro and in vivo following fixed-time artificial insemination

Successful cryopreservation of bison semen is fundamental for restoration of genetic diversity in Canada’s wood bison. Conventional bovine semen extenders contain animal products, such as egg yolk and milk, which are undesirable because of biosecurity risks and undefined composition. In this study, we examined the efficacy of an exogenous protein-free extender containing cholesterol-cyclodextrin complex (CC) to cryopreserve bison semen. The study also provided an opportunity to determine the effectiveness of different ovulation synchronization protocols for fixed-time artificial insemination in bison. Semen was collected from wood bison bulls via electroejaculation and cryopreserved in either Tris-egg yolk-glycerol (called ‘TEYG’) extender or pretreated with CC (2 mg/mL semen) and diluted in Tris-glycerol (collectively called ‘CC-TG’) extender. Post-thaw sperm motion characteristics and in vitro fertilization of cattle oocytes confirmed that CC alone without egg yolk protected bison sperm during cryopreservation process. In the first fertility trial, however, no pregnancy was obtained following fixed-time artificial insemination of bison cows with CC-TG extender. In a follow-up trial, low concentration of CC (1 mg/mL semen) resulted in better post-thaw sperm motion characteristics, fertility rate, and birth of live calves following fixed-time artificial insemination. Results showed that 1 mg CC/mL semen completely replaced egg yolk in bison semen extender. In addition, both follicular ablation and steroid treatment protocols provided ovulation synchrony to permit successful application of fixed-time artificial insemination in bison. This is the first report on the birth of live bison calves following fixed-time artificial insemination using semen cryopreserved in a defined extender.

Correlation between the sperm characteristics evaluated by a computerized system (CASA) and the reproductive performance of sows

Computer-assisted semen analysis (CASA) systems have been one the most used tools to evaluate sperm kinetics. The objective of this research was to estimate the correlation between sperm motility characteristics evaluated by CASA during 72 hours of cooling with the farrowing rate (FR) and total number of piglets born (TNB) after artificial insemination. Multiparous sows (n=464) were inseminated with semen from seven boars (19.6 ± 1.3 ejaculates/male). Sperm motility parameters were determined immediately after dilution and after 24, 48 and 72 hours of cooling at 15°C: total motility (TM-%), progressive motility (PM-%), curvilinear velocity (VCL-μm/s), straight line velocity (VSL-μm/s), average path velocity (VAP-µm/s), amplitude of lateral head displacement (ALH-µm), flagellar beat cross frequency (BCF-Hz), straightness (STR-%) and linearity (LIN-%). Pearson’s correlation coefficient was applied to analyze the data and the comparison of the means of the sperm characteristics between the boars was done by Tukey’s test at 5% probability. TM and PM at time zero (T0) were significant and had a moderate to high correlation with FR and TNB. After 72 hours of refrigeration, the semen quality was reduced and showed a significant and low correlation of the TM and PM with these same parameters. The boar presenting the lowest value of TM and PM after dilution obtained lower FR and TNB. In conclusion, computer-assisted semen analysis soon after dilution can be used to predict fertility of boars.

Does Coenzyme Q10 Exert Antioxidant Effect on Frozen Equine Sperm?

During semen cryopreservation, the sensitivity of equine sperm to oxidative stress is increased by the eliminated seminal plasma. Thus, antioxidant addition to the semen extender can be helpful to the sperm survival after freezing and thawing. This work aimed to test whether coenzyme Q10 (CoQ10) added in different concentrations to the INRA 82 freezing extender has antioxidant function on equine sperm to improve its fertilizing ability. Semen samples from five stallions were frozen with the extenders: (T1) INRA 82, control, (T2) T1+ 5 μM CoQ10, (T3) T1+ 25 μM CoQ10, and (T4) T1+ 50 μM CoQ10. After sample thawing, sperm motility and kinetics characteristics were evaluated using a computer-assisted sperm analysis and sperm membrane functionality and integrity were evaluated with a hypo-osmotic swelling test and an epifluorescence microscopy, respectively. The nitrite (NO2-) and hydrogen peroxide (H2O2) concentrations of the semen samples were measured with spectrophotometry. There was no difference on the sperm characteristics among all treatments (P > .05). However, the 25 μM CoQ10 (T3) decreased NO2− concentration (6.7 ± 2.2 μM/μg protein) compared with the treatments T1, T2, and T4 (64.3 ± 3.7, 59.4 ± 5.3, 45.1 ± 8.6 μM/μg protein), respectively, as well H2O2 concentration (1.8 ± 0.3 μM/μg protein) compared with the control (4.6 ± 0.4 μM/μg protein) and 5 μM CoQ10 treatments (4.8 ± 0.2 μM/μg protein, P < .05). In conclusion, 25 μM CoQ10 plays a significant role as antioxidant to the frozen equine sperm, decreasing NO2− and H2O2 concentrations. Thus, its addition to the INRA 82 freezing extender may be beneficial to the fertilizing ability of equine semen.

Effects of glucose concentration in semen extender and storage temperature on stallion sperm quality following long-term cooled storage

In Experiment 1, the effects of glucose concentration in extender (0 mM, 67 mM, 147 mM, 270 mM; G0, G67, G147, and G270, respectively) and storage temperature of extended semen (5, 10, 15 and 20 °C) were evaluated after storage for up to 5 days (T0h to T120h). For all time points tested, mean total (TMOT) and progressive (PMOT) sperm motility were lower in G0 than all other treatment groups (P < 0.05). Mean curvilinear velocity (VCL) was lower in G0 than other treatment groups at all time points tested except T0h (P < 0.05). Mean percentage of plasma membrane/acrosome intact sperm (VAI) was similar among treatments at T0h, T72h, and T120h (P > 0.05). Mean TMOT and PMOT, were lower for semen stored at 20 °C than all lower storage temperatures (P < 0.05) at all time points. In Experiment 2, semen was stored at 10 °C in extender containing no added glucose (G0) or 147 mM glucose (G147). Following storage, semen was centrifuged and resuspended in extender containing no added glucose (G0 – G0 or G147 – G0, respectively) or 147 mM of glucose (G0 – G147 or G147 – G147, respectively). Mean TMOT, PMOT, and VCL were higher in G147 than G0 at all time periods tested (P < 0.05), whereas mean VAI was similar between these treatment groups throughout the experiment (P > 0.05). Mean TMOT and PMOT were higher in G0 – G147 than G0 – G0 at T72h and T120h (P < 0.05) and mean VCL was higher in G0 – G147 than G0 – G0 for all time periods. Mean TMOT, PMOT, and VCL were higher in G147 – G147 than G147 – G0 at all time points tested (P < 0.05), whereas mean VAI was similar between these two treatment groups for each of the time points (P > 0.05). In Experiment 3, the minimum concentration of glucose required to maintain sperm quality following long-term cooled storage (T120 h) was evaluated (G0, G5, G10, G20, G40, G67, G147 mM). At T120 h, mean TMOT was lowest in G0, G5, G10, and G20 (P < 0.05), whereas mean PMOT and VCL were lower in G0, G5, G10, and G20 than in G40, G67, and G147 (P < 0.05). Mean VAI was higher in G10 than G67, but similar among G10 and other treatment groups (P > 0.05). In conclusion, the absence of added glucose in extender reduced the motion characteristics of stallion sperm during long-term storage (5 days), but VAI was not affected. The use of temperatures between 5 and 15 °C for long-term storage (5 days) best maintained sperm motility and VAI. The threshold concentration of added glucose in extender required to optimize sperm motion characteristics was 40 mM.

Dietary omega-3 fatty acids from linseed oil improve quality of post-thaw but not fresh sperm in Holstein bulls

Docosahexaenoic acid (DHA), a member of the n-3 fatty acid family present in fish oil, has several positive effects on bovine sperm, including membrane integrity, motility and viability, as well as cold sensitivity. Our objective was to investigate effects of varying amounts of omega-3 fatty acids from linseed oil, administered orally, on quality of fresh and frozen-thawed bull sperm. Twenty fertile Holstein bulls (874 ± 45.38 kg) were randomly and equally assigned to four groups and received encapsulated (rumen-protected) fats for 12 weeks, as follows: group P, 300 g palm oil; group Pl, 200 g palm oil + 100 g linseed oil; group pL, 100 g palm oil + 200 g linseed oil; and group L, 300 g linseed oil. Sperm quality of fresh and frozen-thawed semen was evaluated by routine assays including sperm motion characteristics (CASA), membrane integrity (eosin-nigrosin), membrane activity (hypo-osmotic swelling test; HOST) and malondialdehyde (MDA) content. There were no significant differences among groups in semen volume, sperm concentration or sperm quality parameters in fresh semen. However, after freezing-thawing, total and progressive motility in group P (59.61 ± 1.95 and 40.19 ± 2.48%, respectively; LSM ± SEM) were lower (P < 0.05) than in groups Pl (66.06 ± 1.95 and 47.53 ± 2.48%), pL (65.67 ± 1.95 and 47.48 ± 2.48%) and L (65.36 ± 1.95 and 47.62 ± 2.48)%, with no significant differences among the latter three groups. Furthermore, membrane integrity (eosin-nigrosin) and activity (HOST) were lower (P < 0.05) in group P (55.79 ± 2.15 and 42.19 ± 2.17%) compared to groups Pl (62.73 ± 2.15 and 48.93 ± 2.17%), pL (64.06 ± 2.15 and 50.01 ± 2.17%) and L (64.47 ± 2.15 and 49.68 ± 2.17%), with no significant differences among the latter three. Furthermore, there were more (P < 0.05) morphologically abnormal sperm in group P (25.99 ± 1.62%) than in groups Pl, PL and L (21.55 ± 1.62, 21.69 ± 1.62 and 20.90 ± 1.62%). In conclusion, feeding Holstein bulls 100–300 g linseed oil daily improved sperm cryotolerance.

Effect of olive-derived antioxidants (3,4-dihydroxyphenylethanol and 3,4 dihydroxyphenylglycol) on sperm motility and fertility in liquid ram sperm stored at 15°C or 5°C.

The aim of the present study was to evaluate the effect of the addition of two olive oil-derived antioxidants, hydroxytyrosol (3,4-dihydroxyphenylethanol, HT) and 3,4-dihydroxyphenylglycol (DHPG), on ovine semen during liquid storage at 5°C and 15°C. Semen was collected, pooled, diluted and then divided into aliquots supplemented with different concentrations (5μg/ml, 10μg/ml, 50μg/ml and 100μg/ml) of HT, DHPG and a mixture (MIX) of both antioxidants. Sperm motility characteristics were assessed in the different samples at 0, 6, 24, 48, 72 and 96hr after cooling, and a fertility trial was also conducted. The results showed that the antioxidant addition did not significantly improve total and progressive motility in ovine cooled sperm maintained at 15° or 5°C. However, in samples stored at 5°C, LIN (48, 72, 96hr), STR (0hr) and WOB (0, 48, 72, 96hr) values significantly decreased in comparison with control treatment when high antioxidant concentrations were added (MIX100 or HT100). When samples were maintained at 15°C, MIX50 showed significantly higher VCL values than the control treatment after 6hr cooling, and MIX100 showed significantly lower VCL values at 96hr after cooling. According to the artificial insemination trial, no significant differences were observed when antioxidants were added. In conclusion, the use of HT and DHPG showed small impact in sperm motility and fertility was not affected (nor detrimentally nor positively) when insemination was carried out using antioxidant-supplemented liquid sperm.

Cryopreservation of human spermatozoa with penetrating and non-penetrating cryoprotectants

Сryopreserved spermatozoa are widely used in infertility treatment by assisted reproductive technologies. However, the spermatozoa survival rate remains low in patients with oligoastenoteratozoospermia. Therefore the development of effective cryopreservation methods for spermatozoa from pathospermia is relevant. The effectiveness of cryopreservation spermatozoa from oligoastenoteratozoospermia man using penetrating and non-penetrating cryoprotectants was compared. Sperm motility, viability and morphological characteristics were evaluated after cryopreservation with glycerol and polyvinylpyrrolidone. The average number of spermatozoa count in fresh ejaculate was (11.0±0.2) mln/ml. After isolation of active motile fraction the number of cells was (3.8±0.3) mln/ml and (84.3±8.4) % from them were motile (group 3). (78.8±6.6) % of spermatozoa cryopreserved with glycerol (group 1) and (41.4±8.1) % cryopreserved with polyvinylpyrrolidone (group 2) remained active motile. The spermatozoa viability after cryopreservation was (82.1±8.6) % and (89.6±8.6) % in group 1 and 2, respectively. Despite the high rate of spermatozoa survival in group 1 the number of motile cells decreased to (27.3±4.8) % after cryoprotectant removing stage. Morphological analysis revealed that the incidence of spermatozoa head abnormalities was (25.97±2.67), (19.21±2.67) and (20.57±1.19) % in group 1–3, respectively. The differences of spermatozoa midpiece and tail abnormalities in the study groups were statistically insignificant. The use of polyvinylpyrrolidone as a cryoprotectant allows preserving 90 % of survived spermatozoa from oligoastenoteratozoospermia men after freeze/thawing. The set of spermatozoa head, neck and midpiece abnormalities is significantly lower after cryopreservation with polyvinylpyrrolidone compared with routine method with glycerol. Two-stage spermatozoa cryopreservation method with polyvinylpyrrolidone is promising for assisted reproductive technologies since spermatozoa can be used immediately after warming for oocyte fertilization without cryoprotectant removing step.

Effect of melatonin on bovine sperm characteristics and ultrastructure changes following cryopreservation.

The production of reactive oxygen species (ROS) during cryopreservation of semen alters the sperm motion and mobility characteristics, resulting in poor or failure of conception rate after artificial insemination (AI). Melatonin an antioxidant is able to modulate the effect of ROS and prevents spermatozoa by reducing the oxidative stress during freezing process. Eight ejaculates from eight healthy HF bulls diluted with Tris egg yolk glycerol extender were divided into five equal aliquots. The Computer Assisted Semen Analyser (CASA) results showed no significant difference between the control—post‐ thaw samples and melatonin‐treated samples; however, the velocity of spermatozoa with regard to the VAP, VSL showed highest increase in the 0.25 mM MLT‐treated semen followed by 0.1 mM MLT treated semen except for VCL where velocity increased with increase in the concentration of melatonin. The vigour of spermatozoa regard to BCF, STR and LIN recorded highest increase in the 0.25 mM MLT treated semen followed by 0.1 mM MLT‐treated semen except for the ALH where vigour increased with increase in the concentration of melatonin. The electron micrography images illustrated that the addition of 0.1 mM melatonin protected the plasma membrane and acrosome region and maintained the ultrastructure integrity of the cryopreserved spermatozoa when compared to control group, whereas the electron micrography of spermatozoa treated with 0.2 and 0.25 mM melatonin illustrated highest damage to the plasma and acrosome membrane. Thus concluding that inclusion of melatonin to sperm extender can improve the post‐thaw quality of cryopreserved bull spermatozoa.

The Effect of N-N-Dimethylformamide on the Membrane Characteristics of Canine Spermatozoa After Cryopreservation, and its Relationship With Post-Thaw Motility

Some studies have demonstrated that glycerol is superior to amides in preserving sperm motion characteristics of canine sperm; however, little is known about the effect of these cryoprotectants on the membrane characteristics of canine spermatozoa after freezing/thawing. In this study, the effects of using either N-N-dimethylformamide (DMF) or glycerol (GLY) on the integrity and function of the canine sperm, after cryopreservation were determined. We hypothesized that the use of a multiparametric approach for assessing the effect of DMF on the membranes of canine sperm would explain the lower values reported for post-thaw motility. Ejaculates from 12 dogs were collected, split into 2 groups, and frozen using a tris-fructose-citrate-egg yolk-based extender containing either 7% (v/v) GLY or 7% (v/v) DMF. Frozen straws (n = 120) were thawed and analyzed for subjectively-assessed sperm progressive motility, normal morphology, plasma membrane integrity, plasma membrane function (HOST+), acrosome membrane integrity, high mitochondrial membrane potential, and simultaneous assessment of sperm membrane integrity and function by a triple-staining fluorescent procedure. Overall, sperm motility and membrane intactness/function were higher when GLY was used as a cryoprotectant, as compared to DMF (P < .05). A model to explain the variation in progressive motility using the values obtained from the sperm integrity and function parameters was designed. The percent HOST+ sperm and high mitochondrial membrane potential sperm were mostly associated with the changes observed in the progressive motility (r2 = 0.84; P = .043) when either GLY or DMF were used as cryoprotectants. These results may explain the overall reduced sperm quality observed after cryopreservation, as a reflection of sublethal damage sustained by the sperm membranes.

Which is the major trigger in aquatic environment for pearl oyster Pinctada fucata martensii sperm from gonad: Ammonia ion or pH ?

To understand the roles of ammonia ion, pH, and salinity triggering sperm motility, effect of various ammonia ion concentrations (0, 1, 2, 3, 5, 10, 20 mM), external pH (pHe, 7.00, 8.20, 9.00, 9.50) and salinity (20, 25, 31, 35, 40 psμ) on total motile sperm (TM) and swimming movement characteristics (curvilinear velocity - VCL, straight-line velocity - VSL, average path velocity - VAP, beat-cross frequency -BCF) were investigated for Chinese pearl oyster Pinctada fucata martensii, under laboratory condition. The TM and swimming movement characteristics were analyzed by a computer assisted sperm analysis system, based on sperm parameters of invertebrate. Ammonium ion as the major factor in triggering sperm motility, rather than pHe was proved. Sperm motility was less affected by differing salinity solution without ammonium ion. Seawater containing 2 mM or 3 mM ammonium ion were suggested to be used for motility trigged and movement evolution for P. fucata martensii. The results in the present study are instructive for artificial external fertilization, gamete stocks and management in P. fucata martensii.

English

The aim of this study was to compare the embryonic developmental rates in the Southern African cattle breeds. To do this, cryopreserved semen straws from Nguni, Bonsmara, and Boran bulls were thawed at 38&deg;C and evaluated for sperm motility characteristics using Sperm Class Analyser (SCA). The fertilizing ability of frozen/thawed sperm was evaluated by performing artificial insemination (AI) and in vitro fertilization (IVF). For AI, superovulated cows were inseminated with frozen/thawed semen and then further evaluated for embryo development. For IVF, oocytes from the respective cows were retrieved using ovum pickup, and then matured. Following maturation, oocytes were co-incubated with semen for 6 h. In the Nguni breeds, the IVF method of embryo production was mildly superior to the in vivo method at the morula stage while the Bonsmara breed revealed the opposite effect at both the morula and blastocyst stages. In the Boran breed, the IVF method was highly superior with the in vivo method at the 8-cell stage while the opposite effect was observed at the blastocyst stage of embryonic development. This study suggests that the Boran breed is less susceptible to loss of embryonic development as compared to the Nguni and Bonsmara breeds. &nbsp; Key words: Nguni, Bonsmara, Boran, embryo, beef breeds, motility, artificial insemination, in vitro fertilization.

Two-step accelerating freezing protocol yields a better motility, membranes and DNA integrities of thawed ram sperm than three-steps freezing protocols

The present study compares a protocol that mimics freezing of ram semen in static nitrogen vapor with two protocols with an initial low cooling rate in the first step, followed by higher cooling rates where ice nucleation occurs. Semen ejaculates, obtained from twelve adults rams, were diluted with TEST-based extender and frozen with either Protocol 1 (three-step decelerating cooling): from +5 °C to −35 °C (40 °C/min), from −35 °C to −65 °C (17 °C/min), and then from −65 °C to −85 °C (3 °C/min); or Protocol 2 (three-step accelerating cooling): from +5 °C to −5 °C (4 °C/min), from −5 °C to −110 °C (25 °C/min), and then from −110 °C to −140 °C (35 °C/min); or Protocol 3 (two-step accelerating cooling), from +5 °C to −10 °C (5 °C/min), and then from −10 °C to −130 °C (60 °C/min). Post-thaw sperm quality was reduced for all protocols (p < .05) compared with fresh semen. Post-thaw percentages of sperm motility characteristics and sperm with intact plasma membrane, intact acrosome, and intact mitochondrial membrane were greater using Protocol 3 than Protocol 2 (p < .05) and Protocol 1 (p < .01). In addition, the post-thaw percentage of sperm with fragmented DNA was lower (p < .05) using Protocol 3 compared with Protocol 1. The present results indicate that a cooling rate of 60 °C/min around and after the time point of ice nucleation provided better post thaw survival and function of ram sperm than lower (and/or decelerating) cooling rates.

Supplementation of chilling storage medium with glutathione protects rooster sperm quality

This study was aimed to evaluate the effect of addition of reduced glutathione (GSH) to the extender on the rooster's semen quality parameters and fertility potential. Semen samples were diluted with Lake extender contained 0, 0.5, 1, 2, 4 and 8 mM GSH. Then, were chilled to 5 °C and stored for a period of 48 h. Sperm motion characteristics, viability, membrane integrity, lipid peroxidation, mitochondrial activity and fertility potential were evaluated. At the initiation of the experiment (0 h), GSH did not affect sperm parameters, while 2–4 mM GSH improved (P ≤ 0.05) quality indicators during storage periods. Moreover, the samples treated with 2–4 mM GSH have had a lower lipid peroxidation compared to other groups (P ≤ 0.05). Artificial insemination using the semen samples, which had been stored in groups treated with 2–4 mM GSH for a period of 24 h, led to greater (P ≤ 0.05) fertilizing potential compared to the control group.

Effect of post-thaw supplementation of fractionated seminal plasma on survival of boar spermatozoa.

In our recent study we demonstrated that the holding of fresh semen in fractionated seminal plasma (SP1, >40 kDa; SP2, ⟨40 kDa), obtained by gel filtration chromatography, significantly improved the sperm quality characteristics following cryopreservation (Wasilewska-Sakowska et al. 2019). In this study we investigated the effect of post-thaw (PT) supplementation of fractionated SP (SP1 and SP2) on the survival of spermatozoa from boars with good and poor semen freezability, GSF and PSF, respectively. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis showed distinct differences in the protein profiles of SP1 and SP2 from boars with GSF or PSF regarding the number of protein spots. Sperm motility characteristics and the motion patterns, assessed using the computer-assisted sperm analysis (CASA) system, were markedly higher in PT semen supplemented with SP1 and SP2 from boars with GSF. Post-thaw supplementation of either SP1 or SP2 from boars with GSF significantly improved mitochondrial function, plasma membrane and acrosome integrity, and viability during storage. The findings of this study have confirmed that the presence of protective protein components in varying abundance in either fractionated SP from boars with good freezability ejaculates significantly improved the sperm survival following PT storage.

Development of an efficient and standardized method for the cryopreservation of Arctic charr milt and its use in the fertilization of brook trout eggs to produce ‘sparctic’ hybrids

The general objective of this study was to establish a standardized procedure for the cryopreservation of Arctic charr milt and its use in the production of ‘sparctic’ hybrids. The specific objectives of this study were to determine: (i) the optimal final sperm concentration as well as (ii) the final glucose concentration, (iii) the effect of post-thaw storage time on sperm motility characteristics, and (iv) the fertilization ability of cryopreserved Arctic charr milt at 0 and 60 min after thawing at sperm:egg ratios of 500,000:1 and 1,000,000:1. We observed high post-thaw sperm motility (49–57%) at final sperm concentrations ranging from 0.5 to 2.0 × 109 spermatozoa ml−1 in straw. We determined that a final glucose concentration of 0.15–0.19 M in 7.5% methanol was optimal (post-thaw sperm motility 48–60%). Milt cryopreserved at optimal conditions (final concentrations: 0.17 M of glucose, 7.5% methanol and 1.5 × 109 spermatozoa ml−1) could be stored for up to 15 min after thawing without the loss of sperm motility. The ability of cryopreserved milt to fertilize brook trout eggs was high (68–72%) and did not differ between the tested sperm:egg ratios (500,000:1 and 1,000,000:1) when milt was used immediately after thawing or after 60 min of post-thaw storage. The milt used in this study was obtained from two hatcheries and was characterized by significantly different values of sperm concentrations. In conclusion, a standardized and efficient cryopreservation method for Arctic charr milt has been developed and used for the production of ‘sparctic’ hybrids. Species-specific requirements for final sperm and glucose concentrations and the post-thaw storage time were determined. The hatchery of origin of Arctic charr should be taken into consideration, as high variability in sperm characteristics may be expected. The development of this method and the positive results of the fertilization trials indicate promising potential for the implementation of cryopreserved milt in hatchery practice.

195 Sperm motion characteristics of ram semen liquid-stored in a milk egg yolk extender at four temperatures

Abstract Varying temperatures have been used for liquid storage of ram semen and that of other species. This study evaluated motility characteristics of ram semen stored at 5, 10, 15, and 20°C for up to 96 h. Two ejaculates were collected and pooled from each of 6 rams using an artificial vagina and evaluated for motility and concentration. Samples were extended in ultra-high temperature pasteurized skim milk and egg yolk (10% v/v) containing penicillin and streptomycin, diluted to 250 million sperm/mL, and packaged in 0.5 mL straws. Semen was held at 32°C during processing, and straws placed in 500 mL jars for storage at 5°C (refrigerator), 10 and 15°C (refrigerated water bath) and 20°C (air conditioned room temperature). Cooling rates were 0.04, 0.25, 0.44, and 0.02 °C/min, and final temperatures reached after 672, 83, 36, and 567 min in the four storage environments, respectively, with cooling rates faster in a liquid than air environment. Straws from individual rams were removed at 6, 24, 48, 72 and 96 h of storage and analyzed with a computer-assisted sperm analyzer after warming to 36°C. Data were analyzed for the effect of storage time, temperature, and their interaction on sperm motion characteristics. Sperm motion characteristics were not affected over time during storage at 5 and 10°C. At 15°C motility parameters decreased in a curvilinear (P &lt; 0.05) relationship with time (progressive motility: 52.6 to 29.7%; rapid motility: 36.5 to 16.5%), and at 20°C in linear (P &lt; 0.001) relationship (progressive motility: 51.9 to 13.1%; rapid motility: 36.1 to 6.3%). Circular motility decreased (P &lt; 0.05) with increasing temperatures after 72 and 96 h of storage, while local motility was not affected by storage temperature and time. Results suggest storage at 10°C may be a viable alternative to storage at 5°C as retention of motility was similar.

Supplemental Antibiotics in a Commercial Extender for Stallion Semen

Commonly marketed semen extenders contain various antibiotic types and concentrations to control bacterial growth from stallion's external genitalia. An experiment was conducted to test the effects of supplemental amikacin disulfate (1,000 μg/mL) + potassium penicillin G (1,000 IU/ML: INRA-AP), or ticarcillin–clavulanate (1,000 μg/mL: INRA-TIM) in the INRA 96 extender, on sperm function and antimicrobial activity, compared with extender without supplemental antibiotics (INRA-C). In freshly extended semen (Time 30m), no differences were observed among the three treatment groups for sperm motion characteristics or plasma membrane intactness (P > .05). Following cooled storage (Time 24h), sperm progressive motility and straightness were higher in INRA-AP, as compared to INRA-C or INRA-TIM (P < .05). For both time points, INRA-AP yielded lower bacterial colony-forming units (CFU/mL) than INRA-TIM or INRA-C (P < .05). In addition, INRA-AP yielded a higher proportion of culture plates with no growth (59%), than INRA-TIM (14%) or INRA-C (22%; P < .05). These findings suggest that INRA 96 extender can be supplemented with the tested concentrations of amikacin disulfate + potassium penicillin G to improve its antimicrobial effectiveness without impairing sperm quality.

Evaluation of sperm mobility activation media for egg Fertiliztion in locally bred American channel catfish (Ictalurus punctatus)

Fish sperm motility is now thought to be the prime indicator for the peculiarity of fish spermatozoa. Sperm motion characteristics from over 305 different fish species have been published in over 1450 scholarly articles spanning a wide variety of issues, from molecular biology to ecology. The purpose of the current study was to evaluate the impact of two activation media (A, B) created in the lab and distilled water as a control on male American channel catfish milt quality and fertilization rate (Ictalurus punctatus). Brooders were developed from locally bred catfish seed and selected on the basis of maturity at Aquaculture and Fisheries Program, National Agriculture Research Center (NARC), Islamabad Pakistan. The experiment used a complete randomized design (CRD) with three replicates for each of the three treatments: media A, B, and Control (distilled water). As considerable number of sperms are present in every microliter of sperm, hence several forms of activation or dilution media were utilized to utilize sperm or milt more effectively. Following milt collection, it was diluted in a ratio of 1:29 with the aforementioned matching activation media before being subjected to in vitro testing for a number of parameters, including sperm mobility, sperm mobility duration, and sperm viability. The same diluted milt aliquots were used to check fertilization rates, and the findings showed that the components were substantially different in the specified conducts (p

Effect of Fractionated Seminal Plasma on Sperm Characteristics Following Cryopreservation of Boar Semen

Abstract This study aimed to investigate the effect of fractionated seminal plasma (SP) on boar sperm characteristics following cryopreservation. Gel filtration chromatography yielded two fractions: SP1 with more than 40 kDa (&gt;40 kDa) and SP2 with less than 40 kDa (&lt;40 kDa). The fractionated SP (SP1 and SP2), whole seminal plasma (wSP) and Beltsville Thawing Solution (BTS) were used for the treatment of semen before freezing-thawing. Besides the analysis of sperm motility characteristics, plasma membrane integrity (PMI), acrosome integrity, and viability (Vybrant Apoptosis Assay) were analyzed in pre-freeze and post-thaw (PT) semen. Among the analyzed pre-freeze sperm parameters, rapid movement was markedly affected by boar and treatment. Furthermore, boar and treatment were significant sources of variations in PT semen quality. Treatment with wSP caused a marked reduction in PT semen quality compared with BTS, SP1 or SP2. Wide variations in PT acrosome integrity and viability were observed in spermatozoa treated with BTS and the fractionated SP, being significantly higher in the SP1- and SP2-treated samples. However, PT semen quality did not differ between semen samples treated with SP1 and SP2. Representative electrophoretic profiles of sperm proteins from each treatment showed quantitative and qualitative differences, indicating varying effects of the cryopreservation procedure on the sperm membrane integrity. The findings of this study indicated that the cryoprotective effects of the SP components varied among boars and that different components of the fractionated SP exerted varying effects on sperm functions following cryopreservation. It could be suggested that the variable protective protein components of either fractionated SP ameliorated alterations in the sperm membranes during cryopreservation, resulting in reduced susceptibility to cryo-damage.

Effects of CoQ10 on the quality of ram sperm during cryopreservation in plant and animal based extenders

The purpose of this study was to evaluate the effect of different concentrations of CoQ10 in soybean lecithin (SL) or egg yolk (EY) extenders on ram semen cryopreservation. Semen samples were collected from five rams, twice a week, then diluted in the extenders (SL and EY) containing different concentrations of CoQ10 as follows: extender containing SL: 0 μM (control, SL/Q0), 1 μM (SL/Q1), 2 μM (SL/Q2), 5 μM (SL/Q5) and 10 μM (SL/Q10) CoQ10; extender containing EY: 0 μM (control, EY/Q0), 1 μM (EY/Q1), 2 μM (EY/Q2), 5 μM (EY/Q5) and 10 μM (EY/Q10) CoQ10. Sperm motion characteristics, membrane integrity, abnormal morphology, viability, apoptotic-like changes, mitochondria active potential, acrosome integrity and lipid peroxidation were evaluated after freeze-thaw process. The SL/Q1, SL/Q2, EY/Q1 and EY/Q2 resulted in greater (P ≤ 0.05) sperm total motility, progressive motility, membrane integrity and mitochondria active potential compared to the other groups. Acrosome integrity in the SL/Q0, SL/Q1, SL/Q2, EY/Q0, EY/Q1 and EY/Q2 groups was greater (P ≤ 0.05) than in the SL/Q5, SL/Q10, EY/Q5 and EY/Q10 groups. The SL/Q2 and EY/Q2 treatment groups had greater (P ≤ 0.05) sperm viability rates and less apoptotic-like changes and lipid peroxidation. The CoQ10 compound could be explored as a novel potential antioxidant for cryopreservation of ram semen because with used of this compound in the present study there was an improved post-thawed sperm quality.